In humans, one of the commonest of all genetic disorders is autosomal dominant polycystic kidney disease (ADPKD) also termed adult polycystic kidney disease (APKD), affecting approximately 1/1000 individuals (Dalgaard, 1957). ADPKD is a progressive disease of cyst formation and enlargement typically leading to end stage renal disease (ESRD) in late middle age. The major cause of morbidity in ADPKD is progressive renal disease characterized by the formation and enlargement of fluid filled cysts, resulting in grossly enlarged kidneys. Renal function deteriorates as normal tissue is compromised by cystic growth, resulting in end stage renal disease (ESRD) in more than 50% of patients by the age of 60 years (Gabow, et al., 1992). ADPKD accounts for 8-10% of all renal transplantation and dialysis patients in Europe and the USA (Gabow, 1993).
ADPKD also causes cystic growth in other organs (reviewed in Gabow, 1990) and occasionally presents in childhood (Fink, et al., 1993; Zerres, et al., 1993). Extrarenal manifestations include liver cysts (Milutinovic, et al., 1980), and more rarely cysts of the pancreas (Gabow, 1993) and other organs. Intracranial aneurysms occur in approximately 5% of patients and are a significant cause of morbidity and mortality due to subarachnoid haemorrhage (Chapman, et al., 1992). ADPKD is associated with a higher prevalence of various connective tissue disorders. An increased prevalence of heart valve defects (Hossack, et al., 1988), hernia (Gabow, 1990) and colonic diverticulae (Scheff, et al., 1980) have been reported.
Considerable progress has been made in the last few years in understanding the pathophysiology of ADPKD (and other animal models of cystic disease). Cysts in ADPKD are known to develop from outpouchings of descending or ascending kidney tubules and the early stages are characterized by a thickening and disorganization of the basement membrane, accompanied by a de-differentiation of tubular epithelial cells. Several of the characteristics of ADPKD epithelia: altered growth responses, abnormal expression of various proteins and reversal of polarity, may be a sign of this de-differentiation and important in cyst expansion. The nature of the primary defect which triggers these changes is, however, unknown and consequently much effort has been devoted to identifying the causative agent by genetic means.
The first step towards positional cloning of an ADPKD gene was the demonstration of linkage of one locus now designated the polycystic kidney disease 1 (PKD1) locus to the a globin cluster on the short arm of chromosome 16 (Reeders, et al., 1985). Subsequently, families with unlinked to markers on 16p were described (Kimberling, et al., 1988; Romeo, et al., 1988) and a second ADPKD locus (PKD2) has recently been assigned to chromosome region 4q13-q23 (Kimberling, et al., 1993; Peter, et al., 1993). It is estimated that approximately 85% of ADPKD is due to PKD1 (Peters and Sankuijl, 1992) with PKD2 accounting for most of the remainder. PKD2 appears to be milder condition with a later age of onset and ESRD (Parfrey, et al., 1990; Gabow, et al., 1992; Ravine, et al., 1992).
The position of the PKD1 locus was refined to chromosome band 16p13.3 and many markers were isolated from that region (Breuning, et al., 1987; Reeders, et al., 1988; Breuning, et al., 1990; Germino, et al., 1990; Hyland, et al., 1990; Himmelbauer, et al., 1991). Their order, and the position of the PKD1 locus, has been determined by extensive linkage analysis in normal and PKD1 families and by the use of a panel of somatic cell hybrids (Reeders et al., 1988; Breuning, et al., 1990; Germino, et al., 1990). ADPKD is genetically heterogenous with loci mapped not only to 16p13.3 (PKD1), but also to chromosome 4 (DKD2). Although the phenotype of PKD1 and PKD2 are clearly similar, it is now well documented that PKD1 (which accounts for about 85% of ADPKD; (Peters, 1992) is a more severe disease with an average age at ESRD of about 56 years compared to about 71.5 years for PKD2 (Ravine, 1992). An accurate long range restriction map of the 16p13.3 region (Harris, et al., 1990; Germino, et al., 1992) has located the PKD1 locus in an interval of approximately 600 kb between the markers GGG1 and SM7 (Harris, et al., 1991; Somlo, et al., 1992) (see FIG. 1a). The density of CpG islands and identification of many mRNA transcripts indicated that this area is rich in gene sequences. Germino et al. (1992) estimated that the candidate region contains approximately 20 genes.
Identification of the PKD1 gene from within this area has thus proved difficult and other means to pinpoint the disease gene have been sought. Linkage disequilibrium has been demonstrated between PKD1 and the proximal marker VK5, in a Scottish population (Pound, et al., 1992) and between PKD1 and BLu24 (see FIG. 1a), in a Spanish population (Peral, et al., 1994). Studies with additional markers have shown evidence of a common ancestor in a proportion of each population (Peral, et al., 1994; Snarey, et al., 1994), but the association has not precisely positioned the PKD1 locus.
Disease associated genomic rearrangements, detected by cytogenetics or pulsed field gel electrophoresis (PFGE) have been instrumental in the identification of various genes associated with various genetic disorders. Hitherto, no such abnormalities related to PKD1 have been described. This situation contrasts with that for the tuberous sclerosis locus, which lies within 16p13.3 (TSC2). In that case, TSC associated deletions were detected by PFGE within the interval thought to contain the PKD1 gene and their characterisation was a significant step toward the rapid identification of the TSC2 gene (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). The TSC2 gene therefore maps within the candidate region for the hitherto unidentified PKD1 gene; as polycystic kidneys are a feature common to TSC and ADPKD1 (Bernstein and Robbins, 1991) the possibility of an etiological link, as proposed by Kandt et al. (1992), was considered. A contiguous gene syndrome resulting from the disruption of PKD1 and the adjacent tuberous sclerosis 2 (TSC2) gene, which is associated with TSC and severe childhood onset polycystic kidney disease, has also been defined (Brook-Carter, et al. 1994).
We have now identified a pedigree in which the two distinct phenotypes, typical ADPKD or TSC, are seen in different members. In this family, the two individuals with ADPKD are carriers of a balanced chromosome translocation with a breakpoint within 16p13.3. We have located the chromosome 16 translocation breakpoint and a gene disrupted by this rearrangement has been defined; the discovery of additional mutations of that gene in other PKD1 patients shows that we have identified the PKD1 gene. Full characterisation of the PKD1 transcript has been significantly complicated because of the unusual genomic region containing most of the gene. All but 3.5 kb at the 3xe2x80x2 end of the transcript (which is about 14 kb in total) is encoded by a region which is reiterated several times elsewhere on the same chromosome (in 16p13.1 and termed the HG area). The structure of the duplication is complex, with some regions copied more times than others, and the HG region encoding three large transcripts. The transcripts from the HG area are: HG-A (21 kb), HG-B (17 kb) and HG-C (8.5 kb) and although these have 3xe2x80x2 ends which differ from PKD1, over most of their length they share substantial homology to the PKD1 transcript. Consequently, cloning and characterizing a bona fide PKD1 cDNA has proven difficult. To overcome the problem caused by duplication we have cloned cDNAs covering the entire transcript from a cell line which contains the PKD1 but not the HG loci. Characterisation of these cDNAs has enabled the PKD1 protein sequence to be predicted and led to the identification of several homologies with described motifs.
Accordingly, in one aspect, this invention provides an isolated, purified or recombinant nucleic acid sequence comprising:
(a) a PKD1-encoding nucleic acid or its complementary strand,
(b) a sequence substantially homologous to, or capable of hybridizing to, a substantial portion of a molecule defined in (a) above, or
(c) a fragment of a molecule defined in (a) or (b) above.
In particular, there is provided a sequence wherein the PKD1 gene has the nucleic acid sequence according to FIG. 15 (Seq. I.D. No. 7), or the partial sequence of FIGS. 7 (Seq. I.D. No. 1) or 10 (Seq. I.D. No. 5). The invention therefore includes a DNA molecule coding for a polypeptide having the amino acid sequence of FIG. 15 (Seq. I.D. No. 8), or a polypeptide fragment thereof; and genomic DNA corresponding to a molecule as in (a)-(c) above.
As used herein, xe2x80x9csubstantially homologousxe2x80x9d refers to a nucleic acid strand that is sufficiently duplicative of the PKD1 sequence presented in FIG. 15 (Seq. I.D. No. 7) such that it is capable of hybridizing to that sequence under moderately stringent, and preferably stringent conditions, as defined herein below. Preferably, xe2x80x9csubstantially homologousxe2x80x9d refers to a homology of between 97 and 100%. Further, such a strand will encode or be complementary to a strand that encodes PKD1 protein having the biological activity described below. As used herein, a xe2x80x9csubstantial portion of a moleculexe2x80x9d refers to at least 60%, preferably 80% and most preferably 90% of the molecule in terms of its linear residue length or its molecular weight. xe2x80x9cNucleic acidxe2x80x9d refers to both DNA and RNA.
The PKD1 gene described herein is a gene found on human chromosome 16, and the results of studies described herein form the basis for concluding that this PKD1 gene encodes a protein called PKD1 protein which has a role in the prevention or suppression of ADPKD. The PKD1 gene therefore includes the DNA sequences shown in FIG. 15 (Seq. I.D. No. 7), and all functional equivalents. By xe2x80x9cfunctional equivalentsxe2x80x9d, we mean nucleic acid sequences that are substantially homologous to the PKD1 nucleic acid sequence, as presented in FIG. 15 (Seq. I.D. No. 7), and encoding a protein that possesses one or more of the biological functions or activities of PKD1; i.e., that is involved in cell/cell adhesion, cell/cell recognition or cell/cell communication, for example to effect adhesion of cells to other cells or components of the extracellular matrix; effect communication and/or interaction between epithelial cells and the basal membrane (whether in kidneys or otherwise); assist in development of connective tissue such as assembly and/or maintenance of the basal membrane; in signal transduction between cells or cells and components of the extracellular matrix; and/or to promote binding of cells carrying proteins such as integrins or carbohydrates to target cells. The biological function of PKD1 of course includes maintaining a healthy physiological state; that is, the native protein""s aberrations or absence results in ADPKD or an associated disorder.
The PKD1 gene may furthermore include regulatory regions which control the expression of the PKD1 coding sequence, including promoter, enhancer and terminator regions. Other DNA sequences such as introns spliced from the end-product PKD1 RNA transcript are also encompassed. Although work has been carried out in relation to the human gene, the corresponding genetic and functional sequences present in lower animals are also encompassed.
The present invention therefore further provides a PKD1 gene or its complementary strand having the sequence according to FIG. 15 (Seq. I.D. No. 7), which gene or strand is mutated in some ADPKD patients (more specifically, PKD1 patients). Therefore, the invention further provides a nucleic acid sequence comprising a mutant PKD1 gene as described herein, including wherein Intron 43 as defined hereinbelow has a deletion of 18 or 20 bp resulting in an intron of 55 or 57 bp.
As used herein, xe2x80x9cPKD1 mutantxe2x80x9d or xe2x80x9cmutationxe2x80x9d encompasses alterations of the native PKD1 nucleotide (Seq. I.D. No. 7), or amino acid sequence (Seq. I.D. No. 8), as defined by FIG. 15, i.e., substitutions, deletions or additions, and also encompasses deletion of DNA containing the entire PKD1 gene.
The invention further provides a nucleic acid sequence comprising a mutant PKD1 gene, especially one selected from a sequence comprising a partial sequence according to FIGS. 7 (Seq. I.D. No. 1) and/or 10 (Seq. I.D. No. 5), or the corresponding sequences disclosed in FIG. 15 (Seq. I.D. No. 7), when:
(a) [OX114] base pairs 1746-2192 as defined in FIG. 7 (Seq. I.D. No. 1) are deleted (446 bp);
(b) [OX32] base pairs 3696-3831 as defined in FIG. 7 (Seq. I.D. No. 1) are deleted by a splicing defect;
(c) [OX875] about 5.5 kb flanked by the two Xbal sites shown in FIG. 3a are deleted and the EcoR1 site separating the CW10 (41 kb) and JH1 (18 kb) sites is thereby absent
(d) [WS53] about 100 kb extending between the JH1 and CW21 and the SM6 and JH17 sites shown in FIG. 6 and the PKD1 gene is thereby absent, the deletion lying proximally between SM6 and JH17
(e) [461] 18 bp are deleted in the 75 bp intron amplified by the primer pair 3A3C (Seq. I.D. Nos. 11 and 12) insert at position 3696 of the 3xe2x80x2 sequence (Seq. I.D. No. 1) as shown in FIG. 11 (Seq. I.D. No. 18);
(f) [OX1054] 20 bp are deleted in the 75 bp intron amplified by the primer pair 3A3C (Seq. I.D. Nos. 11 and 12) insert at position 3696 of the 3xe2x80x2 sequence (Seq. I.D. No. 1) as shown in FIG. 11 (Seq. I.D. No. 18);
(g) [WS212] about 75 kb are deleted between SM9-CW9 distally and the PKD1 3xe2x80x2UTR proximally as shown in FIG. 12;
(h) [WS-215] about 160 kb are deleted between CW20 and SM6-JH17 as shown in FIG. 12;
(i) [WS-227] about 50 kb are deleted between CW20 and JH11 as shown in FIG. 12;
(j) [WS-219] about 27 kb are deleted between JH1 and JH6 as shown in FIG. 12;
(k) [WS-250] about 160 kb are deleted between CW20 and Blu24 as shown in FIG. 12;
(l) [WS-194] about 65 kb is deleted between CW20 and CW10.
The invention therefore extends to RNA molecules comprising an RNA sequence corresponding to any of the DNA sequences set out above. Such molecule may be the transcript reference PBP and identifiable with respect to the restriction map of FIG. 3a and having a length of about 14 KB.
In another aspect, the invention provides a nucleic acid probe having a sequence as set out above; in particular, this invention extends to a purified nucleic acid probe which hybridizes to at least a portion of the DNA or RNA molecule of any of the preceding sequences. Preferably, the probe includes a label such as a radiolable, for example, a 32P label.
In another aspect, this invention provides a purified DNA or RNA coding for a protein comprising the amino acid sequence of FIG. 15 (Seq. I.D. No. 8), or a protein polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein.
The DNA molecule defined above may be incorporated in a recombinant cloning vector for expressing a protein having the amino acid sequence of FIG. 15 (Seq. I.D. No. 8), or a protein or a polypeptide having at least one functional domain or active site in common with said protein. Such a vector may include any vector for expression in bacteria, e.g., E. coli; yeast, insect, or mammalian cells.
The invention also features a nucleic acid probe for detecting PKD1 nucleic acid comprising 10 consecutive nucleotides as presented in FIG. 15 (Seq. I.D. No. 7). Preferably, the probe may comprise 15, 20, 50, 100, 200, or 300, etc., consecutive nucleotides (nt) presented in FIG. 13, and may fall within the size range 15 nt-13 kb, 100 nt-5 kb, 150 nt-4 kb, 300 nt-2 kb, and 500 nt-1 kb.
Probes are used according to the invention in hybridization reactions to identify PKD1 sequences, whether they be native or mutated PKD1 DNA or RNA, as disclosed herein. Such probes are useful for identifying the PKD1 gene or a mutation thereof, as defined herein.
The invention also features a synthetic polypeptide corresponding in amino acid residue sequence to at least a portion of the sequence of naturally occurring PKD1, and having a molecular weight equal to less than that of the native protein. A synthetic polypeptide of the invention is useful for inducing the production of antibodies specific for the synthetic polypeptide and that bind to naturally occurring PKD1.
Preferred embodiments of this aspect of the invention include a group of synthetic polypeptides whose members correspond to a fragment of the PKD1 protein comprising a stretch of amino acids of at least 8, and preferably 15, 30, 50, or 100 residues in length from the sequence disclosed in FIG. 15 (Seq. I.D. No. 8).
In another aspect, the invention provides a polypeptide encoded by a sequence as set out above, or having the amino acid sequence according to the amino acid sequence of FIG. 15 (Seq. I.D. No. 8), or a protein or polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein. In particular, there is provided an isolated, purified or recombinant polypeptide comprising a PKD1 protein or a mutant or variant thereof or encoded by a sequence set out above or a variant thereof having substantially the same activity as the PKD1 protein. The present invention may further comprise a polypeptide having 9 or 13 transmembrane pairs instead of 11 transmembrane domains as described hereinbelow. Further comprising this invention is a molecule which interacts with a polypeptide as herein described which molecule synergises, causes, enhances or is necessary for the functioning of the PKD1 protein as herein described.
The invention also encompasses recombinant expression vectors comprising a nucleic acid or isolated DNA encoding PKD1 and a process for preparing PKD1 polypeptide, comprising culturing a suitable host cell comprising the vector under conditions suitable for promoting expression of PKD1, and recovering said PKD1.
This invention also provides an in vitro method of determining whether an individual is likely to be affected with tuberous sclerosis, comprising assaying a biological sample from the individual to determine the presence and/or amount of PKD1 protein or polypeptide having the amino acid sequence of FIG. 15 (Seq. I.D. No. 8).
As used herein, xe2x80x9cbiological samplexe2x80x9d includes any fluid or tissue sample from a mammal, preferably a human, including but not limited to blood, urine, saliva, any body organ tissue, cells from any body tissue, including blood cells.
Additionally or alternatively, a sample may be assayed to determine the presence and/or amount of mRNA coding for the protein or polypeptide having the amino acid sequence of FIG. 15 (Seq. I.D. No. 8), or to determine the fragment lengths of fragments of nucleotide sequences coding for the protein or polypeptide of FIG. 15 (Seq. I.D. No. 8), or to detect inactivating mutations in DNA coding for a protein having the amino acid sequence of FIG. 15 (Seq. I.D. No. 8) or a protein having homologous properties. The screening preferably includes applying a nucleic acid amplification process, as described herein in detail, to said sample to amplify a fragment of the DNA sequence. The nucleic acid amplification process advantageously utilizes at least one of the following sets of primers as identified herein: AH3 F9 (Seq. I.D. No. 9), : AH3 B7 (Seq. I.D. No. 10); 3A3 C1 (Seq. I.D. No. 11), : 3A3 C2 (Seq. I.D. No. 12); and AH4 F2(Seq. I.D. No. 13), : JH14 B3 (Seq. I.D. No. 14).
Alternatively, the screening method may comprise digesting the sample DNA to provide EcoRI fragments and hybridizing with a DNA probe which hybridizes to the EcoRI fragment identified (A) in FIG. 3(a), and the DNA probe may comprise the DNA probe CW10 (Seq. I.D. No. 4). identified herein.
Another screening method may comprise digesting the sample to provide BamHI fragments and hybridizing with a DNA probe which hybridizes to the BamHI fragment identified (B) in FIG. 3(a), and the DNA probe may comprise the DNA probe 1A1H.6 identified herein.
A method according to the present invention may comprise detecting a PKD1-associated disorder in a patient suspected of having or having predisposition to the disorder (i.e., a carrier), the method comprising detecting the presence of and/or evaluating the characteristics of PKD1 DNA, PKD1 mRNA and.or PKD1 protein in a sample taken from the patient. Such method may comprise detecting and/or evaluating whether the PKD1 DNA is deleted, missing, mutated, aberrant or not expressing normal PKD1 protein. One way of carrying out such a method comprises: A. taking a biological, tissue or biopsy sample from the patient; B. detecting the presence of and/or evaluating the characteristics of PKD1 DNA, PKD1 mRNA and/or PKD1 protein in the sample to obtain a first set of results; C. comparing the first set of results with a second set of results obtained using the same or similar methodology for an individual that is not suspected of having the disorder; and if the first and second sets of results differ in that the PKD1 DNA is deleted, missing, aberrant, mutated or not expressing PKD1 protein then that is indicative of the presence, predisposition or tendency of the patient to develop the disorder. As used herein, a xe2x80x9cPKD1-associated disorderxe2x80x9d refers to adult polycystic kidney disease, as described herein, and also refers to turberous sclerosis, as well as other disorders having symptoms such as cyst formation in common with these diseases.
A specific method according to the invention comprises extracting from a patient a sample of PKD1 DNA or DNA from the PKD1 locus purporting to be PKD1 DNA, cultivating the sample in vitro and analyzing the resulting protein, and comparing the resulting protein with normal PKD1 protein according to the well-established Protein Truncation Test. Less sensitive tests include analysis of RNA using RT PCR (reverse transcriptase polymerase chain reaction), and examination of genomic DNA.
Step C of the above method may be replaced by: comparing the first set of results with a second set of results obtained using the same or similar methodology in an individual that is known to have the or at least one of the disorder(s); and if the first and second sets of results are substantially identical, this indicates that the PKD1 DNA in the patient is deleted, mutated or not expressing normal PKD1 protein.
The invention further provides a method of characterizing a mutation in a subject suspected of having a mutation in the PKD1 gene, which method comprises: A. amplifying each of the exons in the PKD1 gene of the subject; B. denaturing the complementary strands of the amplified exons; C. diluting the denatured separate, complementary strands to allow each single-stranded DNA molecule to assume a secondary structural confirmation; D. subjecting the DNA molecule to electrophoresis under non-denaturing conditions; E. comparing the electrophoresis pattern of the single-stranded molecule with the electrophoresis pattern of a single-stranded molecule containing the same amplified exon from a control individual which has either a normal or PKD1 heterozygous genotype; and, F. sequencing any amplification product which has an electrophoretic pattern different from the pattern obtained from the DNA of the control individual.
The invention also extends to a diagnostic kit for carrying out a method as set out above, comprising nucleic acid primers for amplifying a fragment of the DNA or RNA sequences defined above, and packaging means therefore. The kit may optionally include written instructions stating that the primers are to be used for detection of disorders associated with the PKD1 gene. The nucleic acid primers may comprise at least one of the following sets: AH3 F9 (Seq. I.D. No. 9): AH3 B7 (Seq. I.D. No. 10): 3A3 C1 (Seq. I.D. No. 11): 3A3 C2 (Seq. I.D. No. 12): and AH4 F2 (Seq. I.D. No. 13): JH14 B3 (Seq. I.D. No. 14).
Another embodiment of kit may combine one or more substances for digesting a sample to provide EcoRI fragments and a DNA probe as previously defined. A further embodiment of kit may comprise one or more substances for digesting a sample to provide BamHI fragments and a DNA probe as previously defined.
A vector (such as Bluescript (available from Stratagene) comprising a nucleic acid sequence set out above; and a host cell (such as E. coli strain SL-1 Blue (available from Stratagene) transfected or transformed with the vector are also provided, together with the use of such a vector or a nucleic acid sequence set out above in gene therapy and/or in the preparation of an agent for treating or preventing a PKD1-associated disorder.
Therefore, there is further provided a method of treating or preventing a PKD1-associated disorder which method comprises administering to a patient in need thereof a functional PKD1 gene to affected cells in a manner that permits expression of PKD1 protein therein and/or a transcript produced from a mutated chromosome (such as the deleted WS-212 chromosome) which is capable of expressing functional-PKD1 protein therein.
As used herein, the term xe2x80x9chybridizationxe2x80x9d refers to conventional DNA/DNA or DNA/RNA hybridization conditions. For example, for a DNA or RNA probe of about 10-50 nucleotides, moderately stringent hybridization conditions are preferred and include 10xc3x97SSC, 5xc3x97Denhardts, 0.1% SDS, at 35-50 degrees for 15 hours; for a probe of about 50-300 nucleotides, xe2x80x9cstringentxe2x80x9d hybridization conditions are preferred and refer to hybridization in 6xc3x97SSC, 5xc3x97Denhardts, 0.1% SDS at 65 degrees for 15 hours.
The present invention further provides the use of PKD1 protein or polycystin or a mutant or variant thereof having substantially the same biological activity there as in therapy. In particular, to effect cell adhesion, recognition or communication for example to effect adhesion of cells to other cells or components of the extracellular matrix; effect communication and/or interaction between epithelial cells and the basal membrane (whether in kidneys or otherwise); assisting in development of connective tissue such as assembly and/or maintenance of the basal membrane; in signal transduction between cells or cells and components of the extracellular matrix; and/or to promote binding of cells carrying proteins such as integrins or carbohydrates to target cells.
Accordingly, where it is preferred to administer the polypeptide directly to a patient in need thereof, the invention further provides the use of a PKD1 protein or polycystin in the preparation of a medicament. Therefore, there is also provided a pharmaceutical formulation comprising a PKD1 protein, functional PKD1 gene and/or a transcript produced from a mutated chromosome which is capable of expressing functional PKD1 protein, in association with a pharmaceutically acceptable carrier therefor.
The invention also features an immunoglobin, i.e., a polyclonal or monoclonal antibody specific for an epitope of PKD1, which epitope is found in the amino acid sequence presented in FIG. 15 (Seq. I.D. No. 8).
The invention also features a method of assaying for the presence of PKD1 in a sample of mammalian, preferably human cells, comprising the steps of: (a) providing an antibody specific for said PKD1; and (b) assaying for the presence of PKD1 by admixing an aliquot from a sample of mammalian cells with antibody under conditions sufficient to allow for formation and detection of an immune complex of PKD1 and the antibody. Such method is useful for detecting disorders involving aberrant expression of the PKD1 gene or processing of the protein, as described herein.
Preferably, this method includes providing a monoclonal antibody specific for an epitope that is antigenically the same, as determined by Western blot assay, ELISA or immunocytochemical staining, and substantially corresponds in amino acid sequence to the amino acid sequence of a portion of PKD1 and having a molecular weight equal to less than that of PKD1.
The invention thus also features a kit for detecting PKD1, the kit including at least one package containing an antibody or idiotype-containing polyamide portion of an antibody raised to a synthetic polypeptide of this invention or to a conjugate of that polypeptide bound to a carrier. An indicating group or label is utilized to indicate the formation of an immune reaction between the antibody and PKD1 when the antibody is admixed with tissue or cells.
Further features will become more fully apparent in the following description of the embodiments of this invention and from the appended claims.